This invention relates to novel inhibitors of retroviral protease. More particularly, the invention is concerned with ketomethylene isosteric replaced amide bond modified peptide inhibitors of retroviral protease such as human immunodeficiency virus (HIV) protease. As such, these inhibitors have potential use for the treatment of acquired immune deficiency syndrome (AIDS) and aids related complex (ARC).
Acquired immune deficiency syndrome, which only a few years ago was a medical curiosity, is now a serious disease. As a consequence, a great effort is being made to develop drugs and vaccines to combat AIDS. The AIDS virus, first identified in 1983, has been described by several names. It is the third known T-lymphocyte virus (HTLV-III) and has the capacity to replicate within cells of the immune system and thereby lead to a profound destruction of T4.sup.+ T-cells (or CD4.sup.+ cells). See, e.g., Gallo et al., Science 224, 500-503 (1984), and Popovic et al., Ibid., 497-500 (1984). This retrovirus has been known as lymphadenopathy-associated virus (LAV) or AIDS-related virus (ARV) and, most recently, as human immunodeficiency virus (HIV). Two distinct AIDS viruses, HIV-1 and HIV-2, have been described. HIV-1 is the virus originally identified in 1983 by Montagnier and co-workers at the Pasteur Institute in Paris [Ann. Virol. Inst. Pasteur 135 E, 119-134 (1984)], while HIV-2 was more recently isolated by Montagnier and his co-workers in 1986 [Nature 326, 662 (1987)]. As used herein HIV is meant to refer to these viruses in a generic sense.
Although the molecular biology of AIDS is beginning to be unraveled and defined, much more needs to be learned and understood about this disease. In the meantime, numerous approaches are being investigated in the search for potential anti-AIDS drugs and vaccines. Development of an AIDS vaccine is hampered by lack of understanding of mechanisms of protective immunity against HIV, the magnitude of genetic variation of the virus, and the lack of effective animal models for HIV infection. See, for example, Koff and Hoth, Science 241, 426-432 (1988).
The first drug to be approved by the U.S. Food and Drug Administration (FDA) for treatment of AIDS was zidovudine, better known under its former name, azidothymidine (AZT). Chemically, this drug is 3'-azido-3'-deoxythymidine. This drug was originally selected as a potential weapon against AIDS because it was shown to inhibit replication of the virus in vitro. Such in vitro tests are useful and virtually the only practical method of initially screening and testing potential anti-AIDS drugs. A serious drawback of AZT, however, is its toxic side-effects. Thus, the search for better anti-AIDS drugs continues.
Another approach being investigated recently for potential use in the treatment of AIDS is the development of synthetic peptides as inhibitors of retroviral protease. Thus, it is known that retroviruses, including the human immunodeficiency virus (HIV), express their genetic content by directing the synthesis of a polyprotein by the host. This precursor is then processed by proteolysis to give essential viral enzymes and structural proteins. A virally encoded enzyme, the retroviral protease, is contained within the polyprotein and is responsible for the specific cleavages of the polyprotein yielding mature viral proteins. See, for example, Krausslich and Wimmer, Ann. Rev. Biochem. 57, 701-754 (1988).
Inhibition of the HIV protease as a means of therapeutic intervention in the treatment of AIDS and ARC patients is a logical strategy. Inhibition of virally encoded proteases as an approach to antiviral therapy has been demonstrated by Korant et al., J. Cell. Biochem. 32, 91-95 (1986), who showed that an endogenous inhibitor of cysteine proteases, cystatin, inhibited replication of poliovirus in tissue culture. A number of observations in the last few years serve to rationalize this approach for HIV. Isolation and sequence analysis of the HIV-1 [Ratner et al., Nature 313, 277-284 (1985)] and HIV-2 [Guyader et al., Nature 326, 662-669 (1987)] viral genomes show homology of a segment [Toh et al., Science 231, 1567 (1986)] with other retroviral proteases [Yasumaga et al., FEBS Lett. 199, 145 1986)] which show limited homology with a known class of proteolytic enzyme, the aspartic proteinases [Rich, "Inhibitors of aspartic proteinases" in Proteinase Inhibitors, Barrett and Salvesen, eds., Elsevier Science Publ., Amsterdam, 1987, pp. 179-217]. This includes the presence of a conserved Asp-Thr-Gly sequence characteristic of the active site of aspartic proteinases. Known inhibitors of this class of enzyme (pepstatin at high concentration and DAN) inhibit the protease activity of avian myeloblastosis virus [Dittmar and Moelling, J. Virology 28, 106 (1978)], bovine leukemia, Maloney murine leukemia and human T-cell leukemia viruses as shown by Dittmar and Moelling, supra. and by Katoh et al., supra. Several groups [Hansen et al., J. Virology 62, 1785-1791 (1988) and Krausslich et al., Ibid. 62, 4393-4397 (1988)] have demonstrated inhibition by pepstatin of processing of gag-pol polyprotein by HIV protease. Nutt et al., Proc. Natl. Acad. Sci. USA 85, 7179-7233 (1988), reported that pepstatin inhibited synthetic HIV protease with a K.sub.i =1.4 .mu.molar. This link to aspartic proteinases presents the opportunity to draw upon the knowledge-base gathered on renin, the most prominent aspartic proteinase, to expedite the development of specific and potent inhibitors of HIV proteases.
The HIV protease has been shown to be essential in the maturation of viral proteins necessary for viral multiplication such as the gag core proteins when expressed in yeast [Kramer et al., Science 231, 1580 (1986)] or the reverse transcriptase (RT) when expressed in E. coli [Farmerie et al., Science 263, 305 (1987)]. Modification by Seelmeier et al., Proc. Natl. Acad. Sci. USA 85, 6612-6616 (1988), and by Mous, et al., J. Virology 62, 1433-1436 (1988), of the aspartic residue (Asp-25) corresponding by sequence homology to the active site residue by site-directed mutagenesis prevents processing of the polyprotein. Kohl et al., Proc. Natl. Acad. Sci. USA 85, 4686-4690 (1988), have also shown in an E. coli expression system that the replacement of Asp-25 with Asn in HIV protease prevents cleavage of gag p55 in cultured cells and inhibits infectivity in tissue culture. Loeb et al., J. Virology 63, 111-121, (1989) have carried out an extensive mutagenesis study on HIV protease expressed in E. coli and shown inhibition of gag processing by both conservative and non-conservative mutation of the amino acids adjacent to the assumed active site. This demonstrates in HIV a similar role for the protease to that shown by Katoh et al., Virology 145, 280-292 (1985), for Maloney murine leukemia virus where deletions in the protease region led to immature virus particles with markedly reduced infectivity.
Modification of known substrate sequences is an accepted approach to inhibitor generation. HIV protease cleaves the virally encoded polyprotein at several sites to liberate the gag proteins (p17,p24,p15) as well as the protease (Prot) itself and reverse transcriptase (p66). Thus, small peptides overlapping each of the cleavage sites (p17/p24, p24/p15, p15/Prot, and Prot/p66) become candidates for modification. Schneider and Kent, Cell 54 363 (1988), have prepared peptide substrates of twenty or more residues corresponding to each of these cleavage sites and demonstrated that their synthetic enzyme cleaved at the appropriate site. Darke et al., Biochem. Biophys. Res. Commun. 156, 297-303 (1988), showed that both expressed and synthetic enzyme cleaved at the proposed processing sites with larger synthetic peptides. Cleavage of VSQNYPIV, corresponding to the cleavage site between gag p17 and gag p24, occurred at the same rate as a fifteen residue substrate with a K.sub.m =2.5 mM. The minimal length peptide substrate that was shown to be cleaved (at 90% of the rate of the octapeptide) was a heptapeptide, SQNY-PIV. Either SQNY-PI, QNY-PIV, or acetyl-QNY-PIV failed to be cleaved. In studies on the protease from the retrovirus, avian sarcoma-leukosis virus (ASLV), Kotler et al, Proc. Natl. Acad. Sci. USA 85, 4185-4189 (1988), examined decapeptide substrates and suggest that the minimal size for cleavage with ASLV protease is longer than six residues. An unspecified hexapeptide corresponding to a sequence of HIV containing a Tyr-Pro cleavage site was reported to be a relatively poor substrate and act as an inhibitor of the cleavage of better substrates of ASLV protease.